Individual replacements of the two Arg residues were shown to inactivate and destabilize subgroup II ECF transporters and to reduce ATPase activity of the R. capsulatus BioMNY biotin transporter. A double replacement in BioN abolished complex formation completely. These findings led us to hypothesize that the cytoplasmic Arg-containing motifs of T components could be contact sites for physical and functional interaction with the cytoplasmic A units. This scenario would resemble in part the organization of classical ABC transporters in which coupling helices of the transmembrane domains interact with a groove in the NBDs formed by residues in and around the Q loop. In the present study, we chose the BioMNY system and analyzed the potential physical interaction of the two Argcontaining motifs in the T unit BioN with the A unit BioM by site-specific crosslinking. Nine mono-cysteine variants of BioN with single Cys residues in and around the ARS and ARG sequences were constructed and co-produced with BioM variants containing single Cys residues in the Q loop and the adjacent helical domain. Among 64 combinations analyzed, 28 gave distinct and pronounced thiol crosslinking products. This indicates that the ARS/ARG-containing region of the T unit and the region adjacent to the Q loop of the A units indeed are in physical contact. Moreover, our observation that all nine monoCys BioN variants are partially crosslinked to give homodimers may indicate an oligomeric arrangement of the T component in BioMNY complexes. Quantitative real-time polymerase chain reaction is the method of choice for nucleic acid sequence detection and quantification. Compared to other methods, the major advantages of qPCR are its high throughput, sensitivity, accuracy, and versatility. Using DNA of known concentrations to create a calibration curve, we can quantify the precise copy number of a specific nucleic acid sequence. The DNA standards used to construct the calibration curve could be plasmid DNA, a PCR amplicon, synthesized oligonucleotide, genomic DNA, or cDNA. Among these DNA standards, plasmid DNA is the most commonly used because it is relatively easy to produce and handle. Due to the quantification of absolute DNA copy number by qPCR is based on a DNA standard curve, any amplification bias or measurement error of the DNA standard will compromise the accuracy of the qPCR analysis. Since qPCR is the gold standard for DNA copy number analysis, any compromise in accuracy will be a major concern for PCR applications such as pathogen detection, food regulation, and scientific research. It has been shown that plasmid DNA directly purified from Escherichia coli exists most often in supercoiled form. However, the supercoiled structure of plasmid DNA is vulnerable to heat, mechanical shear, and freeze-thaw, which are common events in the laboratory. These damages could cause DNA strand breaks, changing the supercoiled plasmid into nicked-circular, closedcircular, or linear forms. Previous reports have shown that the conformation of plasmid DNA can have significant effects on DNA amplification by qPCR. There are 2 major types of DNA quantification methods used in routine molecular biology experiments: UV absorbance and fluorescent dye-binding methods. UV absorbance is a Evofosfamide low-cost, moderately sensitive and reliable method to quantify high quality DNA.